Polyketides are a large family of structurally diverse and complex natural products with a wide range of biological functions. In nature, polyketides are biosynthesized by large polyketide synthase enzymes (PKS) through the successive condensations of simple monomers. The long-term goal of the proposed research is to understand the PKSs in order to generate novel polyketides through bioengineering techniques. For modular PKS, each individual module contains all the necessary active sites to carry on one round of synthesis. The proposed research will express and purify the individual modules of 6 deoxyerythronolide B synthase (DEBS). DEBS is a modular PKS responsible for the biosynthesis of the antibiotic erythromycin. Substrates are designed to specifically probe the underlying rules for binding and processing of different substrates by the individual modules. Native and hybrid bimodular constructs will also be prepared and subjected to the novel substrates to understand the incorporation of the substrates by the first module and transfer to the second module for further chemical transformations. Understanding the substrate specificities of the PKS is an important step towards combinatorial biosynthesis of diverse polyketides.